Escherichia coli is one of the leading causes of hospital-acquired infections. The annual cost of nosocomial infections in the U.S. alone is estimated to be in excess of 7 billion dollars. E. Coli is also the most common cause of community acquired urinary tract infection resulting in an estimated 8 million physician visits per year in the U.S.. Physical and genetic maps will be constructed of two large, approximately 107 kilobase pair (each) elements (Pathogenicity Islands, PAIs) from E. Coli J96. PAIs represent insertions within tRNA genes of uropathogenic E. Coli chromosomes. The PAIs are not commonly found in the genomes of normal fecal E. Coli isolates or E. Coli K-12. The principal hypothesis to be tested is that virulence genes responsible for the pathogenesis of E. Coli extra intestinal infections, such as pyelonephritis and sepsis are linked within PAIs. This hypothesis is based on the general pattern of virulence gene blocks seen in other pathogens and the preliminary data presented in this proposal. DNA sequence analysis of the two PAIs provide significant matches to previously characterized virulence genes and mobile genetic elements recognized in other pathogens, such as Salmonellas, Shigella, Yersinia, enterotoxigenic E. Coli, Vibrio, Haemophilus, Bordetella, Pseudomonas, Serratia and Streptococcus. The DNA sequence analysis is being performed through collaboration with a commercial company, Human Genome Sciences (HGS). The specific aims of this proposal are: 1. to construct J96 allelic knock-outs of at least eight of the potential virulence determinants identified thus far and then test these mutants in murine models of ascending urinary tract infection and peritonitis; 2. to independently assess by signature-tagged transposon mutagenesis and negative selection of avirulent mutants the significance of other PAI-genes and potential virulence genes located elsewhere in the genome for uropathogenic E. Coli; 3. to perform molecular epidemiological investigations of the new virulence genes among pathogenic members of the Enterobacteriaceae. The long term objective of the proposed project is to create a comprehensive data base of virulence genes for E. Coli involved in serious human diseases. This information will be of use for the development of new chemotherapeutic and vaccine strategies.